Effects of combined nitrification inhibitors on soil nitrification, maize yield and nitrogen use efficiency in three agricultural soils

Application of nitrification inhibitors (NIs) with nitrogen (N) fertilizer is one of the most efficient ways to improve nitrogen use efficiency (NUE). To fully understand the efficiency of NIs with N fertilizer on soil nitrification, yield and NUE of maize (Zea mays L.), an outdoor pot experiment with different NIs in three soils with different pH was conducted. Five treatments were established: no fertilizer (Control); ammonium sulfate (AS); ammonium sulfate + 3, 4-dimethyl-pyrazolate phosphate (DMPP) (AD); ammonium sulfate + nitrogen protectant (N-GD) (AN); ammonium sulfate + 3, 4-dimethyl-pyrazolate phosphate + nitrogen protectant (ADN). The results showed that NIs treatments (AD, AN and ADN) significantly reduced soil nitrification in the brown and red soil, especially in AD and ADN, which decreased apparent nitrification rate by 28% - 44% (P < 0.05). All NIs treatments significantly increased yield and NUE of maize in three soils, especially ADN in the cinnamon soil and AD in the red soil were more efficiency, which significantly increased maize yield and apparent nitrogen recovery by 5.07 and 6.81 times, 4.39 and 8.16 times, respectively. No significant difference on maize yield was found in the brown soil, but AN significantly increased apparent nitrogen recovery by 70%. Given that the effect of NIs on both soil nitrification and NUE of maize, DMPP+N-GD was more efficient in the cinnamon soil, while N-GD and DMPP was the most efficiency in the brown and red soil, respectively. In addition, soil pH and soil organic matter play important role in the efficiency of NIs.

The result showed that NIs treatments (AD, AN and ADN) significantly reduced soil nitrification in 31 the brown and red soil, especially in AD and ADN, which decreased apparent nitrification rate 28% 32 -44% (P < 0.05). All NIs treatments significantly increased yield and NUE of maize in three soils, 33 especially ADN in the cinnamon soil and AD in the red soil were more efficiency, which 34 significantly increased maize yield and apparent nitrogen recovery by 5.07 times and 6.81 times, 35 4.39 and 8.16 times, respectively. No significant difference on maize yield was found in the brown 36 soil, but AN significantly increased apparent nitrogen recovery by 70%. In conclusion, all NIs 37 treatments performed better in inhibiting nitrification in the brown soil than in the cinnamon and 38 red soil. DMPP and N-GD were more effective in the red soil than in the cinnamon soil. DMPP + 39 N-GD was the most efficient in improving maize yield in the brown soil than other two soils, while 40 DMPP performed better in improving apparent nitrogen recovery in the red soil than other two soils. 41 Introduction 45 Nitrogen (N) fertilizers can increase food production by almost 50%, which made a 46 significantly contribution in alleviating the global food shortage [1]. Hence, tons of N fertilizers are 47 applied to obtain high yield of grain. However, plants are rarely able to absorb more than 50% of 48 the N fertilizer applied to cropping systems, and most of them are lost through NO3leaching, NH3 49 volatilization and N2O emission excessive application of N fertilizers, which caused many problems, 50 such as economical loss, lower nitrogen use efficiency (NUE) and environment pollution [2]. It is 51 necessary, therefore, to find out an efficient way to improve NUE, increase crop yield and mitigate 52 environmental pollution. 53 Adding nitrification inhibitors (NIs) into ammonium-based fertilizer is one of the considerably 54 effective technologies that can inhibit nitrification, reduce N loss, thus improving crop yield and 55 NUE in agricultural systems [3-4]. Nitrification is a major process impacting N cycling in the high-56 production agricultural systems [5]. NIs are compounds that delay the process of nitrification of 57 NH4 + to NO3and subsequent by depressing the activities of nitrifiers and denitrifiers in soil [6].  Values indicate mean ± standard deviations (n = 3). TC, total soil carbon; N, total soil nitrogen; 119 NH4 + -N, ammonium nitrogen; NO3 --N, nitrate nitrogen; P, phosphorus; K, potassium. triple superphosphate and potassium chloride were applied with an application rate of 0.3g N, 0.12 125 g P2O5 and 0.15 g K2O per kg soil, respectively. The application rates of DMPP and N-GD were 126 0.5% and 1.5%, respectively on the w/w basis of N, and the application rate of every single NI was 127 reduced by 50% in NIs combinations treatment. All the amendments were basal dressed, and air-128 dried soil (equivalent to 8kg of oven-dry weight) was thoroughly mixed with the corresponding 129 amendments before added a plastic pot of a 28-cm diameter with a 26 cm-height. Five seeds were 130 sown in each pot, and the seedlings were thinned to one per pot after germination and seedling 131 establishment. Soil moisture content was adjusted daily with deionized water to 60% of the 132 comparisons among the treatments were further explained using Duncan test. Differences were 165 considered significant at P < 0.05. All statistical analyses were performed using SPSS Version 22.0. 166 Graphs were prepared with Origin 9.0. 167 169 We clearly observed that the application of N fertilizer and NIs significantly increased the soil 170 inorganic N content in all three agricultural soils (Fig 1). However, there was no significant 171 differences in soil NH4 + -N content of all treatments at the later stages of maize growth, which 172 decreased about 10 mg kg -1 , 11 mg kg -1 and 24 mg kg -1 in the brown soil, cinnamon soil and red soil, 173 respectively (Fig 1A, C, E, P > 0.05). There was the same pattern between of NO3 --N content with 174 the NH4 + -N content, which increased at the seedling stage and gradually declined followed the 175 growth of maize (Fig 1B, D, F). In the brown soil, the NH4 + -N content in AD was the highest than 176 that in other treatments at the seedling stage ( Fig 1A, P < 0.05). No significant differences were 177 detected between AN and ADN for NH4 + -N content (Fig 1A, P > 0.05). At the elongation stage, the 178 NH4 + -N content in AD and AN was higher compared with ADN ( Fig 1A). We also found that AN 179 maintained higher NH4 + -N content until the maturity stage of maize. All treatments with NIs 180 significantly decreased NO3 --N content at the seedling stage, especially AN was lower in NO3 --N 181 content at the later stages (Fig 1B).  In the cinnamon soil, the addition of other NIs significantly increased soil inorganic N expect 188 for AN. AD maintained higher NH4 + -N content for a long time, while ADN had higher NO3 --N 189 content during the growth of maize (Fig 1C, P < 0.05). In addition, ADN had higher inorganic 190 nitrogen than AN (Fig 1D). 191

NH4 + -N content and NO3 --N content
In the red soil, all treatments with NIs had significantly increased NH4 + -N content compared 192 with AS at the seedling stage (Fig 1E, P < 0.05). In addition, ADN can maintain higher NH4 + -N 193 content for a long period. Lower NO3 --N values in AD and ADN were found compared with AS,194 while ADN had no significantly difference in NO3 --N content with AS (Fig 1F, P >0.05). 195

Apparent nitrification rate
196 Apparent nitrification rate (ANR) indicates that intensify of soil nitrification. Apparent 197 nitrification rate was higher in the cinnamon soil than that in the other two soils (Fig 2). In the brown 198 soil, all treatments with NIs had significantly lower ANR over AS during the whole stage of maize 199 growth. AD was the lowest at the seedling stage, followed by AN (Fig 2A). In the cinnamon soil, 200 higher ANR was observed in treatments of adding NIs into N fertilizer, especially in ADN, which 201 showed that NIs significantly increased soil inorganic nitrogen (Fig 2B). In the red soil, the lowest 202 values were observed in both AD and ADN (Fig 2C, P < 0.05). In addition, ADN had significantly 203 decreased nitrification during the whole growth stage of maize (Fig 2C). In general, NIs with ammonium sulfate significantly increased aboveground biomass 211 compared to AS in all the tested soils (Fig 3, P < 0.05). Grain yield of maize was significantly 212 affected by both soil types and NIs (Table 2, P < 0.01). Both grain and straw biomass had the highest 213 value in the brown soil than those in the cinnamon soil and red soil (Fig 3). In the brown soil, grain 214 yield was the highest in AN followed by AD and AN, which were significantly higher than AS (Fig  215   3A, P < 0.05). No significant differences was detected in the application of NIs with ammonium 216 sulfate (Fig 3A, P > 0.05). 217  F value, the ratio of mean squares of two independents samples; 227 *** Indicates significance at P < 0.001. 228 In the cinnamon soil, the highest value of grain yield was found in ADN than that in other 229 treatments (Fig 3B, P < 0.05). AN resulted in significant increase in grain yield when compared to 230 AS (P < 0.05), while there was no significant difference between AD and AS for grain yield of 231 maize (P > 0.05). 232 In the red soil, the grain yield in NIs treatments was higher compared with AS (Fig 3C, P <  233 0.05). The highest value was found in AD in comparison to AN and ADN (Fig 3C). 234

Nitrogen uptake of maize 235
In the three agricultural soils, all treatments with NIs increased N uptake by maize significantly 236 compared with control (Fig 4, P < 0.05). The highest value of grain and total nitrogen uptake of 237 maize was found in the cinnamon soil (Fig 4). In the brown soil, there was no significant difference 238 between AD and AN, which had significantly higher in N uptake of maize than other treatments 239  In the cinnamon soil, the highest N uptake of maize values were detected in ADN, which 247 significantly higher than other treatments (Fig 4B, P < 0.05). AN had higher N uptake of maize than 248 that of AS, while AD had no significant differences from AS (Fig 4B, P < 0.05). 249 In the red soil, NIs with N fertilizer had significantly increased N uptake of maize compared 250 with AS. AD significantly increased N uptake of maize compared with other treatments (Fig 4C, P  251 < 0.05), while ADN was better than AN (Fig 4C). In the cinnamon soil, the highest values in both ANUE and AR were from ADN, which had 269 significantly different from other treatments. AN was higher in NUE than AD and AS. AD in AR 270 was significantly higher than AS, while ANUE from AD had no significant differences from AS 271 treatment (Fig 5B, P > 0.05). 272 In the red soil, AD had the highest value in both ANUE and AR among all treatments, which 273 had significantly different with the other treatments (Fig 5C, P < 0.05). No significant differences 274 in ANUE were found between AN and ADN (Fig 5C, P >  It is worth to mention that nitrification in red soil becomes stronger, which was according with Lu 281 et al [31], mainly due to the tillage during the decades. In the present study, NIs treatments 282 considerably reduced soil nitrification for all three soils, especially in both brown soil and red soil, 283 a result which was mainly driven by lower pH and fertility. The efficacy of NIs in inhibiting 284 nitrification was reduced because of the rapid hydrolysis of NIs at high soil pH [32]. 285 The effect of NIs varied among different types of soil. NIs significantly increased soil NH4 + 286 and NO3content in both soils expect for cinnamon soil, which was consistent with Guo et al [33], 287 who found that DCD with urine had the potential to increase NH4 + content and maintain it higher 288 for a long time. However, NIs can reduce ANR in all three soils, which was line in with Gong et al 289 [34], who found that DCD and DMPP with urea significantly suppressed potential nitrification rate. 290 In our study, DMPP significantly inhibited nitrification in all soils. DMPP is indiscriminately 291 binding and interaction with ammonium monooxygenase to inhibit the first rate-limiting step of soil 292 nitrification [35]. In addition, DMPP is a heterocyclic N compound with the advantages of low 293 mobility, slow biodegradation and persistence [36]. We also found that at the later stage of maize, 294 NH4 + -N content was gradually declined, which was in line with Zaman et al [12].The main reasons 295 were the decomposition of NIs, N uptake of maize, ammonia volatilization and microbial 296 immobilization [37][38]. 297

298
There are many studies to test the effect of NIs on crops production and N uptake, but it is 299 difficult to draw general conclusions because the performance of the options varied across sites due experiment also showed that the application of DMPP increased 4.2%-4.7% and 6.6%-7.5% of rice 307 and rape grain yield, respectively [39], which was in line with our study. NIs can maintain higher 308 NH4 + -N content for a long period, which improve crop more available N absorption [40]. 309 Ammonium ions usually adsorb by clay particles or soil organic matter, thus decreasing N loss [41]. In terms of cost, the price of DMPP is so expensive that its wide application is limited [41]. In 318 the present study, we found that DMPP had the similar efficiency with N-GD in the brown soil, 319 while in the cinnamon soil DMPP+N-GD was more efficient than DMPP alone, thus our best choice 320 is the application of N-GD and DMPP+N-GD in brown soil and cinnamon soil, respectively. 321

Effects of soil properties on NIs use 322
In our study, the efficiency of NIs differed in different agricultural soils, mainly due to the 323 different pH (brown soil: 5.3; cinnamon soil: 7.9; red soil: 4.6) and SOM. Soil pH has been 324 considered as one of the most important factors affecting the availability of NIs, because pH has 325 potential to effect the mobility and degradation rate of the NIs in soils [47]. In our study, the 326 efficiency of DMPP was more stable in lower pH soil than in alkaline soil. DMPP also performs 327 better in neutral than in alkaline soil [48], which was similar to our results. Additionally, the apparent 328 nitrification rate in lower pH soils was lower than in alkaline soils, which is in accordance with the 329 result of Lu et al [31]. In addition to soil pH, the effectiveness of NIs also influenced by SOM. SOM 330 can absorb NIs and provide energy source for the microorganisms, which leads to the degradation 331 of NIs, decreases the ability of NIs to inhibit nitrification [49]. The results was similarly to our study, 332 SOM was higher in cinnamon soil than both brown and red soil, thus the efficiency of DMPP in 333 cinnamon soil was lower than the other two soils. But the effect of DMPP+N-GD had little 334 influenced by SOM. 335 In addition, there is less study of N-GD, so more research should be required to understand its 336 effect and mechanism. Moreover, the effect of NIs on the N cycle in a range of soil types, cultivated 337 vegetation types, and climatic condition should be fully elucidated. 338

339
In our study, we clearly observed NIs with ammonium sulfate can significantly influence soil 340 inorganic nitrogen, improve yield and NUE. DMPP and N-GD had the same efficiency of decreasing 341 soil nitrification, improving yield and NUE of maize in the brown soil, while NIs combination 342 DMPP+N-GD was the best way to increase soil available nitrogen and improving NUE in the 343 cinnamon soils, DMPP was the most efficiency in lowering soil nitrification and increasing maize 344 yield and NUE in the red soil. The study shows that the effect of different NIs varied among different 345 soils, and soil pH is the main factor. 346